KELOMPOK 2

NAMA KELOMPOK:
- AGUNG MULYAWAN
- ANGGITA OKTAVIA PUTRI
- LIA NUR CHOLIFAH
- LINDA NURYANI AZIZ
- MIFTAHUL JANNAH
- NIKI ANNISAA FITRI
- NINA AI RENI
- PANDU ADJI PRAKOSO
- RAPIKA ASRI
- RHESMA ANANTA PUTRI
- YUNI PRIHATININGRUM
KELAS: 4L
SIGNALING PATHWAY OF
CHOLINERGIC RESEPTOR
 KLASIFIKASI RESEPTOR BERDASARKAN SINYAL
TRANSDUKSI
1. Reseptor Ligand-Gated Ion Channel / Ion-channel linked receptors
Disebut juga reseptor ionotropik. Reseptor membran yang langsung terhubung oleh
suatu kanal ion dan memperantarai aksi sinaptik yg cepat. Cth. Reseptor
asetilkolin nikotinik, reseptor GABAa dan reseptor Glutamat.
Ligand (obat) berinteraksi dg reseptor >> signal >> konformasi reseptor >> kanal ion
terbuka >> ion masuk >>depolarisasi / hiperpolarisasi

2. G-Protein Coupled Receptors/ Reseptor Yg tergandeng dg Protein G

Merupakan reseptor membran yangg tergandeng sistem efektor yangg disebut protein
G. Disebut juga reseptor metabotropic.Reseptor 7 transmembran , karena rangkaian
peptida reseptor ini melintasi membran sebanyak 7 kali. Memperantarai aksi yg
lambat beberapa neurotransmitter dan hormone. Cth. Reseptor asetilkolin
muskarinik, adrenergik, dopaminergik dan serotonin.
Transmisi Sinyal melewati membran sel terjadi dlm 4 tahap :.
• Ikatan ligand (obat) dg reseptor.
• Reseptor mengaktifkan G-protein.
• G-protein yg aktif akan mengaktifkan enzim tertentu atau mempengaruhi kanal
ion tertentu.
• Aktivasi enzim menyebabkan perubahan konsentrasi “ second messenger”.
3. Tyrosine Kinase-Linked Receptors/ Reseptor yg terkait aktivitas Kinase
Merupakan reseptor single transmembran. Memiliki aktivitas kinase dlm
signal transduksinya. Cth. Reseptor sitokin, reseptor growth factor,
reseptor insulin.
Mekanisme :
 Obat atau hormon mengikat ‘extracellular domain’.
 Allosteric effect… autofosforilasi pada ‘intracellular domain’.
 ‘intracellular domain’ yg telah mengalami fosforilasi selanjutnya akan
memfosforilasi protein substrat.

4. Ligand-Activated Transcription Factors / Intracellular Receptors

Reseptor ini berada di dalam sitoplasmik atau nukleus. Aksinya

langsung mengatur transkripsi gen yg menentukan sintesis protein
tertentu. Cth. Reseptor steroid, reseptor estrogen, reseptor
PPARγ (Peroxisome Proliferators-Activated Receptor)
Mekanisme :
 ü Cytosolic receptors. Steroid hormon menembus membran sel dan
mengikat reseptor di sitoplasma. Kompleks ligand-reseptor ditranspor
masuk ke nukleus dan berikatan dg rantai DNA untuk meregulasi
transkripsi gen.
 ü Nuclear receptors. Thyroid hormon masuk ke dalam sel dan secara
pasif masuk ke nukleus untuk berikatan dengan reseptornya.
Reseptor kolinergik terbagi 2 type :
 Reseptor ACh Nikotinik

 Reseptor ACh Muskarinik

Banyak dijumpai sistem saraf otonom di perifer

maupun di pusat.
Keduanya berbeda dlm hal transduksi sinyalnya.
 RESEPTOR ACH NIKOTINIK
 Reseptor terkait dengan kanal ion.
 Dapat berikatan dengan nikotin, tetapi juga memiliki
beberapa ikatan dg senyawa lain.
 Berlokasi di neuromuscular junction, ganglia otonom,
medula adrenal, dan susunan saraf pusat. Paling
banyak di neuromuscular junction
 neuromuscular junction adalah sinaps yg terjadi
antara saraf moyorik dengan serabut otot.
 Memperantarai terjadinya kontraksi otot polos.
 RESEPTOR ACH MUSKARINIK
 Mampu mengikat muskarin, suatu senyawa yg berasal dr jamur
Amanita muscaria
 Terdistribusi luas di seluruh tubuh dan mendukung berbagai fungsi
vital, di otak, sistim saraf otonom, terutama saraf parasimpatis.
 Aktivasi reseptor pd perifer : berkurangnya frekuensi denyut jantung,
relaksasi pembuluh darah, konstriksi sal pernafasan, peningkatan
sekresi dr kelenj keringat dan lakrimasi, konstriksi pd otot spinkter
bola mata dan otot siliar mata.
 Di otak : dijumpai pd cerebral kortex, striatum, hippocampus,
thalamus dan brainstem.
 Berpartisipasi dalam banyak fungsi penting, belajar, ingatan dan
kontrol postur tubuh.
 Reseptor tergandeng protein G.
 Terdiri dari 5 subtype : M1, M2, M3, M4, M5
 Reseptor M1, M3, dan M5 tergandeng dengan protein Gq
 Reseptor M2 dan M4 tergandeng dengan protein Gi dan dengan suatu
kanal ion.
 Respons yg timbul dari aktivasi reseptor muskarinik oleh ACh dapat
berbeda, tergantung pada subtipe reseptor dan lokasinya.
 Reseptor Asetilkolin Muskarinik

M1 M2 M3 M4 M5

Distribusi Cortex, Janutng, CNS, Kelenjar Neostriatum Substantia

hipotalamus, otot polos eksokrin, (otak) nigra (otak),
ganglia saluran cerna, mata
simpatik, otot polos,otak,
kelenjar saliva mata

G protein Gq GI Gq GI Gq
terkait

Respon Aktivasi PLC Inhibisi adenilat Aktivasi PLC Inhibisi adenilat Aktivasi PLC
itraseluler
siklase siklase

Contoh peranan Berperan dalam Mengatur denyut Mengatur motilitas Mengatur Mengatur
dalam sistem
bilogis fungsi kognitif dan jantung, suhu GI, sekresi analgesia, mgk pelepasan
memori, stimulasi tubuh, kontrol kelenjar (salivation mengatur dopamin, regulasi
sekresi asam gerakan, analgesia lacrimation), pelepasan dopamin dilatasi pembuluh
lamung kontriksi otot polos darah otak
bronkus
MUSCARINIC RECEPTOR
SIGNALING IN COLON CANCER
 MUSCARINIC RECEPTORS
Muscarinic receptors are members of the large family of
G-protein coupled receptors (GPCR) that activate 5′-
phosphate (G) proteins. G-proteins regulate a wide-range
of biological processes by modulating the activity of
adenylyl cyclase, phosphatidylinositol lipid turnover and
ion channels. Like other G-protein coupled receptors,
muscarinic receptors have seven transmembrane helical
domains connected by three extracellular and three
intracellular loops. Five receptor subtypes, CHRM1-5,
identified by molecular cloning techniques, modulate cell
function by different post-receptor signaling pathways.
Activation of CHRM1, CHRM3 and CHRM5 results in
phospholipid turnover and changes in cell calcium
concentration. Activation of CHRM2 and CHRM4 results
in inhibition of adenylyl cyclase and reduced levels of
cAMP.
 Muscarinic receptors are long-established as
instrumental in neuronal signaling. In addition to the
GI tract, muscarinic receptors are expressed normally
throughout the body, including the brain, eye, heart,
vasculature, lung, bladder and uterus. More recently,
novel observations demonstrated muscarinic receptor
expression and activation in various cancers including
those arising in the brain, breast, colon, skin, lung
and prostate.
 In the GI tract primarily CRHM1, CRHM2 and
CRHM3 are expressed. Gastric acid secretion by
parietal cells is regulated by CHRM3 and pepsinogen
secretion from chief cells is regulated by a mixture of
CHRM1 and CHRM3. In normal colonic epithelium
both CHRM1 and CHRM3 are expressed, while in
colon cancer upregulation of CHRM3 expression is
suggested.
MUSCARINIC RECEPTOR SIGNALING IN
COLON CANCER
 In vitro studies of human colon cancer cell lines
revealed the principal pathways of muscarinic
receptor signaling in colon cancer. More
recently, in vivo murine models of colonic
neoplasia confirmed the importance of
muscarinic signaling in colon cancer.
 FIGURE 1.

Muscarinic signaling in colonic epithelial cells. Acetylcholine (ACh) and secondary bile acids
(BA) activate extracellular muscarinic receptors (CHRM3). Activated CHRM3 stimulates
matrix metalloproteinase-7 (MMP7) to cleave heparin binding epidermal growth factor (HB-
EGF) from Pro-HB-EGF. HB-EGF transactivates epidermal growth factor receptors (EGFR)
resulting in intracellular signaling via both the MEK/ERK and PI3K/AKT signaling
pathways. Phosphorylation of ERK and AKT are shown (P) and promote translocation of
ERK and NF-κB from the cytosol into the nucleus. Resultant gene transcription promotes
cell proliferation and cell survival (inhibition of apoptosis), both hallmarks of neoplasia.
THERAPEUTIC TARGETS OF MUSCARINIC
RECEPTOR SIGNALING IN COLON CANCER
The complexity of the muscarinic signaling cascade in
colon cancer provides many potential targets for
therapeutic intervention. Reducing muscarinic
receptor ligands by altering colon bile acid
composition (e.g., administration of low-dose
ursodeoxycholic acid) was shown to prevent colon
cancer in patients with inflammatory bowel disease,
and may have a role after onset of colonic malignancy.
Agents that decrease ACh production (e.g., choline
acetyltransferase inhibitors) could attenuate the pro-
proliferative effects of muscarinic signaling, but
unless delivered locally would likely have
unacceptable toxicity given the importance of ACh in
neuronal signaling. As described above, muscarinic
receptor antagonists can decrease intestinal neoplasia
in a murine model of colon cancer; the role for these
agents in human colon cancer should be explored.
STIMULATION OF PROTEIN
SECRETION BY
PARASYMPATHETIC NERVES
STIMULATION OF PROTEIN SECRETION BY PARASYMPATHETIC
NERVES
 Neurotransmitters released from parasympathetic
nerves are potent stimuli of lacrimal gland secretion.
In the lacrimal gland, these nerves contain ACh and
VIP that activate two distinct intracellular signaling
pathways. ACh binds to M3 muscarinic receptors
present on basal and lateral membranes of acinar
cells. The activated M3 muscarinic receptor is coupled
to the G protein, Gαq/11. The G protein, in turns,
activates phospholipase C (PLC). PLC hydrolyzes
phosphatidylinositol bisphosphate (PIP2), a membrane
phospholipid, into two second messengers, 1,4,5-
inositol trisphosphate (1,4,5-IP3) and diacylglycerol
(DAG).1,4,5-IP3 binds to IP3 receptors on the
endoplasmic reticulum to release its Ca2+ stores,
increasing the intracellular concentration of calcium,
or [Ca2+]i.
In the lacrimal gland, cholinergic agonists increase 1,4,5-
IP3 within seconds resulting in a rapid increase in [Ca2+]i.
When the intracellular Ca2+ stores are depleted, a
feedback mechanism is activated whereby Ca2+ enters the
cells through Ca2+ channels in the plasma membrane of
the acinar cell. This Ca2+ influx is termed “stored operated
Ca2+ entry” or “capacitative Ca2+ influx” and is responsible
for the slower, sustained phase of increased [Ca2+]i.
Therefore, the cholinergic agonist-stimulated
Ca2+ response is a biphasic response of a rapid burst
released from intracellular stores followed by movement
of Ca2+ into the cell that is sustained until the stimulus is
removed or decreases slowly over time. In conjunction
with calmodulin, the Ca2+ phosphorylates and therefore
activates proteins, as of yet unidentified, that lead to
protein secretion. The increase in intracellular calcium is
necessary for both muscarinic and adrenergic agonist-
evoked exocytosis from acinar cells, and may even be
sufficient to induce exocytosis in the absence of agonist
stimulation.
 FIGURE 2.

Signal transduction pathways activated by cholinergic agonists in the lacrimal gland. ACh,
acetylcholine; M3, muscarinic receptor subtype 3; Gαq, alpha subunit of Gq G protein; PLC,
phospholipase C; Pyk2 and Src, nonreceptor tyrosine kinases; raf, mitogen-activated protein
kinase kinase kinase; MEK, mitogen-activated protein kinase kinase; MAPK, p44/p42
mitogen-activated protein kinase; IP3, inositol trisphosphate; PKC, protein kinase C; ER,
endoplasmic reticulum. (Modified from Dartt DA:Regulation of lacrimal gland secretion by
neurotransmitters and the EGF family of growth factors. Exp Eye Res 73:741, 2001, with
permission.)
 DAG, which is also produced on hydrolysis of PIP2, binds to
and activates several members of the protein kinase C (PKC)
family. The PKC family includes 10 different isoforms and has
been divided into three groups depending on cofactor
requirements. Classic PKCs include PKCα, -βI, -βII, and -γ and
require calcium and phospholipids for activation. Novel PKC
isoforms include PKCδ, ε, η, and θ, and are calcium
independent but phospholipid dependent. Atypical PKC
isoforms are PKCλ and ζ. These isoforms are calcium and
phospholipid independent. The lacrimal gland contains at least
four PKC isoforms, PKCα, δ, ε, and λ. PKD, also known as
PKCμ, is also present in the lacrimal gland. In general, PKC
isoforms have cell-specific localizations and functions, and the
lacrimal gland is no exception. In the lacrimal gland, PKCα
was found to be located in the cytoplasm and plasma
membranes of acinar cells. PKCδ was found in the cytoplasm
of acinar and myoepithelial cells. PKCε was located in the
cytoplasm, basal, and lateral membranes, and on an apical
membrane system that is similar to actin networks of acinar
cells. PKCε was also seen occasionally on myoepithelial cells.
PKCλ was observed in acinar cells on an endomembrane
system, potentially the endoplasmic reticulum or Golgi
apparatus.
 Translocation of PKC from the cytosol to membrane
fractions can be an indication of activation.
Stimulation of lacrimal gland acini with cholinergic
agonists for less than 1 minute translocates PKCα
from the cytosol to the basal and lateral
membranes. Cholinergic agonists also translocate
PKCδ and ε, albeit with a longer time course. With
specific inhibitors of the α, δ, and ε isoforms of PKC,
cholinergic agonists were shown to use all three
isoforms to varying degrees to stimulate secretion.
Inhibition of PKCα reduced cholinergic agonist-
induced protein secretion approximately 75%, whereas
inhibition of PKCε reduced cholinergic agonist-induced
secretion by approximately 50%. Inhibition of PKCδ
reduced cholinergic agonist-induced secretion by
approximately 20%.
 These findings were confirmed by down-regulating
PKC using phorbol esters. Down-regulation with
phorbol esters decreased the amount of PKCα by 79%,
PKCδ by 53%, and PKCε by 10%. This treatment also
decreased cholinergic agonist-stimulated protein
secretion more than 90%. Thus PKCα, δ, and ε play
differential, but overlapping, roles in cholinergic
agonist-stimulated protein secretion.
In summary, cholinergic agonists bind to
M3 muscarinic receptors to activate PLC. PLC
hydrolyses PIP2 to produce 1,4,5-IP3 and DAG. 1,4,5-
IP3 increases [Ca2+]i, whereas DAG activates PKCα, δ,
and ε. Thus, activation of protein secretion stimulated
by cholinergic agonists uses at least three PKC
isoforms along with Ca2+ to stimulate protein
secretion.
THE ROLE OF M3-MUSCARINIC
RECEPTOR SIGNALING IN
INSULIN SECRETION
Insulin secretion by β cells of the islets of Langerhans
in the pancreas is a process tightly regulated by
glucose and other circulating nutrients. It is also
modulated by many other factors, including hormones
and neurotransmitters. One of the most prominent of
these regulatory mechanisms is mediated by
acetycholine (Ach) originating from the
parasympathetic cholinergic input. Although
cholinergic regulation of insulin release has been
known for many years, the mechanism of regulation
and in particular the identity of the subtype of
cholinergic receptor responsible for this regulation
has only recently been established. There are five
cholingeric muscarinic receptor subtypes (M1–M5) and
the work of Gautam and colleagues using transgenic
and gene knockout technology have determined that
the M3-muscarinic receptor (M3R) is the bona fide
acetylcholine receptor that is responsible for
enhancing glucose-dependent insulin release in β
cells.3
 The mechanism by which M3R regulates insulin release was thought to be
primarily via G-protein depending signaling to the calcium and PKC pathways.
As a prototypical Gq/11-coupled receptor, activation of M3R induces the
hydrolysis of membrane phospholipid phosphatidylinositol-4,5-biphosphate
(PIP2), catalyzed by phospholipase C (PLC). This generates two second
messengers, inositol-1,4,5-trisphosphate (IP3) and diacylglycerol (DAG). IP3 in
turn mobilizes calcium from the IP3-sensitive stores while DAG activates PKC.
Both of these pathways have been thought to play an important role in M3R-
mediated insulin secretion.
 However, recent molecular and genetic studies have pointed to further
mechanisms. In particular, sustained insulin release associated with the enteric
phase appears to be mediated by a process that is independent of G-protein
signaling. This is evidenced by studies from our laboratory and others which
demonstrated that protein kinase D1 (PKD1) as one of the key components by
which M3R regulates glucose-dependent insulin release. PKD1 is activated by
the phosphorylated form of the M3R via a G-protein-independent, β-arrestin-
dependent process that results in secretary vesicle priming. In addition, M3R has
also been shown to stimulate insulin release by inhibiting the mitogen activated
protein kinase p38δ activity, which has inhibitory effects on PKD1 in β cells.
 Other studies have, however, returned to the importance of calcium
signaling. Healy and colleagues demonstrated that the expression level
of IP3-receptors in β cells is crucial for M3R-mediated insulin
release.Binding of the adaptor protein ankyrin-B to the IP3-receptors in
β cells stabilizes the receptors and thus enhances the calcium signal in
the cells. Pancreatic islets from heterozygous ankyrin-B mutant
(ankB+/−) mice exhibited a reduction in both basal and carbachol-
stimulated intracellular calcium release, suggesting that the IP3-receptor
is stabilized in the open state.
 In addition, the sodium channel designated NALCN, which is short for
sodium leak channel non-selective, has also been demonstrated to play a
role in M3R-mediated insulin release. This channel, formerly named
Rb21 then VGCNL1, belongs to the four domain ion channel family.
M3R has been shown to activate this channel in the model pancreatic β
cell line, MIN-6, via the Src family of tyrosine kinases (SFKs). In
addition, one more piece of the jigsaw puzzle is the regulators of G-
protein signaling protein, RGS4. Ruiz de Azua and colleagues have
demonstrated that RGS4 negatively modulate M3R-mediated insulin
secretion in β cells due to its selective inhibition of M3R signaling in β
cells.
 Hence, there appears to be a number of possible in vivo mechanisms
that act in concert to regulate the early and late phases of insulin
secretion by M3R
FIGURE 3.

The possible mechanisms of M3-muscarinic receptor (M3R)-mediated insulin

secretion. Conventional studies have provided evidence that the transient, early phase of
insulin secretion is mediated by M3R via G protein-dependent signaling that results in
increase in intracellular calcium and activation of protein kinase C. Recent studies have
shown that the sustained, late phase of insulin secretion is enhanced by M3R via G
protein-independent pathways which requires receptor phosphorylation/arrestin-
dependent signaling to PKD1 and Src family tyrosine kinases signaling to sodium
channel NALCN.
TERIMA KASIH